Method and kit for direct isothermal sequencing of nucleic acids

ABSTRACT

Direct determination of the sequence of an RNA sample is performed under isothermal conditions. An RNA sample containing a target nucleic acid is combined in a single reaction vessel with a reaction mixture containing two polynucleotide primers, a first primer that specifically hybridizes with a target sequence near the 3′ end of the target nucleic acid, and a second primer that specifically hybridizes to the 3′ end of an antisense copy of the target nucleic acid. At least one of the primers is labeled with a detectable label, and at least one of the first or second primer has an RNA polymerase transcription initiation signal at its 5′ end, which signal does not specifically hybridize to the RNA target. The reaction mixture also contains ribonucleotide triphosphates for RNA synthesis, deoxyribonucleotide triphosphates for DNA synthesis, at least one type of dideoxynucleotide triphosphate chain-terminator, and enzymes with the activity of reverse transcriptase, RNAse H, RNA Polymerase and a low discrimination DNA Polymerase such as Thermo Sequenase™. The combined reactants are incubated under isothermal conditions for a length of time sufficient to generate chain-terminated reaction products, and the chain-termninated reaction products are then detected after electrophoretic separation.

This is a national phase application under 35 U.S.C. 371 based onPCT/CA97/00848, which claims priority from US provisional applicationNo. 60/031,257 filed Nov. 12, 1996.

BACKGROUND OF THE INVENTION

This invention is directed towards a method and kit for determining thenucleotide base sequence of a nucleic acid, particularly for use inroutine clinical diagnostic procedures.

DNA sequence-based diagnosis has the potential to become a routineclinical diagnostic test, and is already available in several formats.See, for example, U.S. Pat. Nos. 5,545,527, 5,550,020 and 5,552,283which are incorporated herein by reference. To fully realize thepotential for DNA sequence-based diagnostics, however, the developmentof simplified, and preferably single-tube sequencing techniques will beimportant.

DNA sequencing technique are generally well known. Such sequencing isgenerally performed using techniques based on the “chain termination”method described by Sanger et al., Proc. Nat'l Acad. Sci. (USA) 74(12):5463-5467 (1977). Basically, in this process, DNA to be tested isisolated, rendered single stranded, and placed into four vessels. Ineach vessel are the necessary components to replicate the DNA strand,i.e., a template-dependant DNA polymerase, a short primer moleculecomplementary to a known region of the DNA to be sequenced, andindividual nucleotide triphosphates in a buffer conducive tohybridization between the primer and the DNA to be sequenced and chainextension of the hybridized primer. In addition, each vessel contains asmall quantity of one type of dideoxynucleotide triphosphate, e.g.dideoxyadenosine triphosphate(ddA).

In each vessel, each piece of the isolated DNA is hybridized with aprimer. The primers are then extended, one base at a time to form a newnucleic acid polymer complementary to the isolated pieces of DNA. When adideoxynucleotide is incorporated into the extending polymer, thisterminates the polymer strand and prevents it from being furtherextended. Accordingly, in each vessel, a set of extended polymers ofspecific lengths are formed which are indicative of the positions of thenucleotide corresponding to the dideoxynucleic acid in that vessel.These sets of polymers are then evaluated using gel electrophoresis todetermine the sequence.

Improvements to the original technique described by Sanger et al. haveincluded improvements to the enzyme used to extend the primer chain. Forexample, Tabor et al. have described enzymes such as T7 DNA polymerasewhich have increased processivity, and increased levels of incorporationof dideoxynucleotides. (See U.S. Pat. No. 4,795,699 and EP-A1-0 655 506,which are incorporated herein by reference). More recently, Reeve et al.have described a thermostable enzyme preparation, called ThermoSequenase™, with many of the properties of T7 DNA polymerase. Nature376: 796-797 (1995). The literature supplied with the Thermo Sequenase™product suggests dividing a DNA sample containing 0.5-2 mg of singlestranded DNA (or 0.5 to 5 mg of double stranded DNA) into four aliquots,and combining each aliquot with the Thermo Sequenase™ enzymepreparation, one dideoxynucleotide termination mixture containing oneddNTP and all four dNTP's; and a dye-labeled primer which will hybridizeto the DNA to be sequenced. The mixture is placed in a thermocycler andrun for 20-30 cycles of annealing, extension and denaturation to producemeasurable amounts of dye-labeled extension products of varying lengthswhich are then evaluated by gel electrophoresis.

DNA sequencing can be performed using these procedures on genomic DNA orcDNA (a DNA copy of mRNA). Alternatively, the direct sequencing of mRNAis known using techniques similar to DNA sequencing.

When low levels of substrate template are present, it is generallynecessary to amplify its amount before sequencing reactions can bereliably performed. A well known method of amplifying a DNA strand is bythe polymerase chain reaction (“PCR”). PCR methods are disclosed in U.S.Pat. Nos. 4,683,194, 4,683,195 and 4,683,202, which are incorporatedherein by reference. RNA amplification may be effectively performedusing techniques disclosed in U.S. Pat. Nos. 5,130,238, 5,409,818,5,554,517 (See also Sooknanan, R., van Gemen, B., and Malek, L. T.“Nucleic Acid Sequence Based Amplification” in Molecular Methods forVirus Detection chp. 12 (Academic Press; 1995)), also incorporatedherein by reference. A related RNA amplification method is disclosed byGen-Probe patents WO 9101384, WO 9525180, WO 9503430, U.S. Pat. No.5,399,491, incorporated herein by reference.

The instant invention discloses a simplified method of determining thesequence of a nucleic acid in a patient sample, in a one-pot or singletube sequencing reaction that does not rely on the PCR method.

It is an object of the invention to provide a method and kit fordetermining the nucleic acid sequence of an RNA molecule in an RNAsample obtained from a patient sample.

SUMMARY OF THE INVENTION

The method of the invention permits determination of the sequence ofnucleotides in a target nucleic acid molecule under isothermalconditions. In accordance with the invention, an RNA sample containing atarget nucleic acid is combined in a single reaction vessel with areaction mixture containing first and second polynucleotide primers,wherein the first primer specifically hybridizes with a target sequencenear the 3′ end of the target nucleic acid, and the second primerspecifically hybridizes to the 3′ end of an antisense copy of the targetnucleic acid, and wherein at least the first or second primer has adetectable label, and wherein at least one of the first or second primerhas an RNA polymerase transcription initiation signal at its 5′ endwhich signal does not specifically hybridize to the RNA target,ribonucleosides ATP, GTP, CTP and UTP or their analogues for RNAsynthesis, deoxyribonucleosides dATP, dGTP, dCTP and dTTP or theiranalogues for DNA synthesis, at least one type of dideoxynucleosidechain terminating nucleoside, or its analogue, and enzymes with theactivity of reverse transcriptase, RNAse H, RNA Polymerase andThermoSequenase. The combined reactants are incubated under isothermalconditions for a length of time sufficient to generate chain-terminatedreaction products, and the chain terminated reaction products are thendetected after electrophoretic separation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a first embodiment of the method of the inventionschematically;

FIG. 2 shows a second embodiment of the method of the inventionschematically; and

FIG. 3 shows a third embodiment of the method of the inventionschematically.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for determining the nucleic acidsequence of an RNA molecule in an RNA sample obtained from a patientsample. The RNA molecule may be human or from an animal (particularlymammalian or avian) or may be from a pathogen such as a virus orbacteria which has contaminated the human or animal sample. Suitablepatient samples include any tissue, blood, other organs, hair follicles,tumor samples extracted by biopsy or otherwise, or an excretioncontaining cells such as urine or sputum.

The RNA sample is prepared according to known methods, such as thepreferred method for the NASBA reaction disclosed in Sooknanan et al.,i.e., by silica pelleting. Care must be taken to prevent thecontamination of the sample by the operator or by aerosol contaminants.

In a first embodiment of the invention, the nucleic acid sequence of theRNA molecule (the “target molecule”) in the prepared RNA sample isdetermined by adding the RNA sample to a reaction mixture containing thefollowing components:

(a) First Primer: a polynucleotide primer of 5-50 nt length which iscapable of specific hybridization with a target sequence near the 3′ endof the target molecule. This primer may be optionally labeled with afirst detectable label, such as a fluorescent (e.g. Cy 5.5, FITC etc.)or radioactive moiety. This primer may also optionally have an RNAPolymerase promoter sequence at its 5′ end, which sequence does nothybridize specifically with the target molecule.

(b) Second Primer: a polynucleotide primer of 5-50 nt length which iscapable of specific hybridization with an anti-sense copy of the targetsequence, near the 3′ end of the anti-sense molecule. This primer may beoptionally labeled with a second detectable label, different from thefirst detectable label of the first primer, such as a fluorescent orradioactive moiety. This primer may also optionally have an RNAPolymerase promoter sequence at its 5′ end, which sequence does nothybridize specifically with the anti-sense copy of the target molecule.

(c) Reverse Transcriptase: a molecule, such as avian myeloblastosisvirus (AMV) reverse transcriptase (Seikagaku, Rockville, Md.), with atleast an RNA directed DNA Polymerase activity. This enzyme generates acDNA copy of an RNA molecule.

(d) RNAse H: an enzyme which selectively degrades RNA in a DNA/RNAhybrid molecule. This activity may be included in the reversetranscriptase enzyme.

(e) Low Discrimination DNA Polymerase: a DNA directed DNA polymerasewith a reduced ability to distinguish between dideoxyribonucleotides(ddNTPs) and deoxyribonucleotides (dNTPs), thus tending to incorporatechain-terminating ddNTPs in primer extension reactions. Preferredenzymes incorporate dideoxynucleotides into an extending nucleic acidpolymer at a rate which is no less than about 0.4 times the rate ofincorporation of deoxynucleotides. Examples of specific enzymes includeThermoSequenase(TM) (or related enzymes such as Sequenase 2.0) (AmershamLife Sciences, Cleveland, Ohio). This enzyme need not necessarily beheat stable since it is being used in an isothermal reaction.

(f) RNA Polymerase: an RNA polymerase such as T7 RNA Pol or T3 RNA Polwhich recognizes the RNA Pol promoter sequence of the first or secondprimer.

(g) Ribonucleotide triphosphates (NTPs) and deoxyribonucleotidetriphosphates (dNTPs): for nucleotide chain polymer synthesis (4 of eachrepresenting nucleotide bases A, C, G and T (or U for RNA)).

(h) At least one chain-terminating dideoxynucleotide triphosphate(ddNTP) for termination of chain extension at a selected base.

The above components are mixed in an appropriate buffer, in appropriateconcentrations, to constitute the reaction mixture.

When the RNA sample is mixed with the reaction mixture and incubated,chain-terminated reaction products are formed which can be analyzed todetermine the sequence of the initial sample. While not intending to bebound by any particular mechanism, these products are believed to beformed in the steps shown in FIG. 1.

First, as shown in step (a), the first primer hybridizes with the 3′ endof the target molecule. Non-specific hybridization is minimized byselection of a suitably high temperature for the reaction (35-65 degreesC.). In step (b), primer extension begins with reverse transcriptaseusing dNTP monomers. Once the chain is extended, (c) the RNAse Hactivity will selectively degrade the original target molecule, leavinga single anti-sense DNA strand. (d) The second primer now hybridizeswith the 3′ end of the anti-sense DNA strand. Primer extension begins(e) with the DNA directed DNA polymerase, such as ThermoSequenase, anddNTP monomers are incorporated into the extending chain. Simultaneously,if the second primer has the non-hybridizing RNA promoter sequence, a“fill in” chain extension will add additional nucleotides onto theanti-sense DNA.

Because of the presence of the at least one ddNTP, two reaction productsmay result from the ThermoSequenase reaction. (f) If no ddNTP isincorporated in the nucleotide chain, a full length cDNA may begenerated. This reaction product can serve as a template for synthesisof the target molecule by RNA Pol using NTP monomers, given that thesecond primer has an RNA Pol promoter sequence. Newly synthesized RNAcan then join the reaction sequence at step (a). RNA Pol will producevery large numbers of RNA transcripts under isothermal conditions from asingle cDNA (5 to 1000 copies), thus substantially amplifying the amountof target sequence.

The second reaction product (g) is the periodic chain terminationproduct that results from incorporation of a ddNTP. Because the secondprimer has a detectable label on it, when the reaction products areseparated by electrophoresis or otherwise (mass spectrometry, etc.),they can be detected and used for determining nucleotide sequence, inthe Sanger et al. method.

It should be noted that even though a chain terminated product has anRNA Pol promoter on it (h), an RNA generated from it will not containthe first primer hybridization site, and will not, therefore, beamplified at the cDNA level.

From this discussion, it is evident that the proper dNTP:ddNTP rationeeds to be ascertained, to generate a suitable amount of full lengthvs. chain terminated fragments. In the method of the invention, thisratio is determined to fall within the range 1:300 to 5000:1, preferablyabout 1000:1. The ratio of dNTP to ddNTP is the same for each type ofnucleotide, A, C, G or T.

The invention illustrated in FIG. 1 may be further improved by severaladditional enhancements. One such enhancement is illustrated in FIG. 2.This method relies on the observation that certain reverse transcriptase(“RT”) enzymes such as Moloney Murine Leukemia Virus (MMLV)RT and MMLVRT have an inherent RNAse H activity (c.f. patent no. EP 408295). Thispermits the reaction to proceed from step (b) to step (c) in the absenceof RNAse H itself, as long as the RT has this RNAse H activity.

Another enhancement, illustrated in FIG. 3 employs the finding,disclosed in EP 655506, that the ability of a DNA polymerase todistinguish between ddNTPs and dNTPs is influenced by a limited numberof amino acids in the active site (ddNTP binding site) of the enzyme.This suggests that a reverse transcriptase could be engineered which hasa reduced ability to distinguish between ddNTPs and dNTPs.

Wild type reverse transcriptase incorporates ddNTPs into a chainextending nucleotide rather poorly. For this reason, negligible amountsof ddNTPs are incorporated into the RT reaction (step (b) of FIG. 1),especially if the dNTP:ddNTP ratio is at the preferred concentration of1000:1.

In fact, wild type reverse transcriptase will incorporate ddNTPs into achain extending strand at a rate of 1 per 10 to 50 dNTPs, which issomewhat greater than wild type Taq Polymerase, E. coli DNA Pol I or T4DNA Pol, where the incorporation rate is less than 1 per 1000. (SeeSooknanan, page 284; or EP 655506). Remarkably, it is found that dideoxyresistant mutant DNA polymerases can be generated by modifying thesingle amino acid that corresponds to position 526 of T7 DNA Pol,position 762 of E. coli Pol 1 and 667 of Taq DNA polymerase. Suchchanges can provide a 250-8000 fold reduction in discrimination levels.Further, modification of at least 13 other sites in the molecule canreduce discrimination although the effect of these alterations is muchless, only 5-20 fold.

Dideoxy resistant reverse transcriptase can be generated by sitespecific mutagenesis techniques. These techniques are well known in theart, (see Sarkar and Sommer 8 Biotechniques 404, 1990) and are explainedin detail as they relate to DNA polymerases in EP 655506. Briefly, thetechnique involves the cloning and expression of mutant forms of a geneencoding a wild type enzyme. Mutations may be introduced either randomlyor by site specific techniques. Expressed mutants are then assayed forddNTP incorporation rates. Presumably, mutations corresponding to thedideoxyresistance mutations observed in other enzymes will be sufficientto create a dideoxy resistant reverse transcriptase. A series ofexperiments can be performed by anyone skilled in the art which would bereasonably expected to generate a dideoxy-resistant mutant of reversetranscriptase.

With such a non-discriminating RT, the method of the invention can beadvanced. In FIG. 3 the RT chain extension at step (b) is the source ofboth the chain terminated fragments and full length fragments. The DNAdirected DNA polymerase employed at step (d) is a high fidelity DNApolymerase, which is highly selective against ddNTP incorporation, suchas E. coli DNA Pol I or Taq Polymerase. In this case, the first primerhas the detectable label. The RT step (b) generates chain terminatedfragments. Any fragments which are fully extended and not chainterminated are used to generate the second strand of the cDNA (d,e). TheRNA template generated in step (f) feeds back into the cycle and canserve as the template for chain termination reactions. This method hasthe advantage that the RNA is the sequencing template, and it does notneed to be converted into cDNA for chain termination reactions. Loweramounts of dNTPs can be usefully employed.

Practice of the method of the present invention can be facilitated bypackaging the various enzymes and reagents used in the invention in kitform. For any given target molecule, such a kit includes:

(a) First Primer: a polynucleotide primer of 5-50 nt length which iscapable of specific hybridization with a target sequence near the 3′ endof the target molecule. This primer may be optionally labeled with afirst detectable label, such as a fluorescent (e.g. Cy 5.5, FITC etc.)or radioactive moiety. This primer may also optionally have an RNAPolymerase promoter sequence at its 5′ end, which sequence does nothybridize specifically with the target molecule.

(b) Second Primer: a polynucleotide primer of 5-50 nt length which iscapable of specific hybridization with an anti-sense copy of the targetsequence, near the 3′ end of the anti-sense molecule. This primer may beoptionally labeled with a second detectable label, different from thefirst detectable label of the first primer, such as a fluorescent orradioactive moiety. This primer may also optionally have an RNAPolymerase promoter sequence at its 5′ end, which sequence does nothybridize specifically with the anti-sense copy of the target molecule.

(c) Enzymes providing reverse transcriptase activity, RNAse H activity,RNA Polymerase activity and DNA polymerase activity having reduceddiscrimination between dNTP and ddNTP substrates. The enzymes may beprovided in individual packages or as a premixed composition containingall of the enzymes activities.

The kit may further contain Ribonucleosides (NTPs) anddeoxyribonucleosides (dNTPs), one or more chain-terminatingdideoxynucleoside (ddNTP) for termination of chain extension at aselected base, and appropriate buffers.

The following examples explain how to perform the method of theinvention and to achieve its intended results. These examples are notintended to limit the scope of the invention in any way.

EXAMPLE 1 Preparation of RNA Sample

An RNA sample containing virus HIV-1 is prepared by adding 0.5-1.0 mlsample (serum of plasma) to a 15-ml conical screw cap tube containing9.0 ml lysis buffer (120 g GuSCN, 2.6 g Triton X-100, 100 ml L2 buffer(12.1 g/l Tris-HCl pH6.4), 22 ml 0.2M EDTA, pH 8.0, final volume 222ml). Invert tubes to mix. Add 70 ul silica suspension and vortex tubefor 5 sec. Leave to 10 1 min at room temp (18-25 deg. C.). Invert everyminute to mix. Centrifuge at 1500 g for 2 min. Remove supernatant usinga 10 ml plastic pipette, leaving about 0.5 ml residual fluid. Removeresidual fluid with sterile pipette without disturbing the pellet. Add 1ml wash buffer (120 GuSCN, 100 ml L2 buffer) and resuspend silica byvortexing. Transfer silica suspension to a 1.5 ml microfuge tube.Centrifuge at 10,000 g for 15 sec. Remove supernatant with sterilepipette. Wash the silica pellet four times, one with wash buffer, twicewith 70% ethanol and once with acetone.

Dry silica pellet completely by placing the opened tube in a heatingblock at 56 deg. C. for 10 1 min. Cover the tube with tissue to avoidaerosol contamination. Add 100 ul elution buffer (0.211 g Tris-HCl, pH8.5/l, sterile) or water and resuspend pellet by vortexing. Incubate at56 deg. C. for 10 1 min to elute the nucleic acid. Centrifuge at 10,000g for 2 min. Transfer supernatant to a new microfuge tube withoutdisturbing the pellet. Store at −70 deg. C.

EXAMPLE 2 Direct Isothermal Sequencing

From stock solutions prepare the following reaction mixture in anuclease free microfuge tube.

Component Concentration Volume buffer 2.5 X 10 ul Dithiothreitol 250 mM1 ul Primer mixture 4 X 6.25 ul Nuclease-free Water to 18 ul

buffer 2.5×: 100 mM Tris, pH 8.5, 125 mM KCl, 30 mM MgCl2, 2.5 mM eachdNTP (dATP, dGTP, dCTP, dTTP), 5 mM each NTP (ATP, GTP, CTP, UTP), 10 uMone or more chain terminating dideoxynucleoside (ddATP, ddCTP, ddGTP,ddTTP)

Primer Mixture: 5 pmol first primer, 5 pmol second primer, 3.75 ul 100%DMSO, Water (nuclease free) to 6.25 ul.

First Primer 5′-AGTGGGGGGACATCAAGCAGCCATGCAAA-3′ SEQ ID No. 1

Second Primer5′-AATTCTAATACGACTCACTATAGGG-TGCTATGTCACTTCCCCTTGGTTCTCTCA-3′ SEQ ID No.2

These sequences to gag gene mRNA of HIV-1. The second primer ischimeric, the first section being a T7 RNA Polymerase promoter.

The second primer is labeled on its 5′ end. The label selected dependson the detection apparatus to be employed. For use with a MicroGeneBlasterÔ (Visible Genetics Inc, Toronto, Canada) a suitable label is thefluorescent dye Cy5.5 (Amersham Life Sciences, Cleveland, Ohio)conjugated to the 5′ terminal nucleotide of the primer, by a dye-esterlinkage.

Vortex the reaction mixture briefly and aliquot 18 ul into a nucleasefree 1.5 ml microfuge tube. Add 5 ul RNA sample and mix by tapping.Incubate at 65 deg. C for 5 min. Transfer to a 40 1 deg. C. water bath,and equilibrate for 5 mins.

Add 2 ul Enzyme Mixture* to the reaction tubes and gently mix bytapping. Centrifuge at 10,000 g for 5 sec. Incubate at 40 1 deg. C. for90 mins. Centrifuge briefly to collect condensate. Place on ice untilready for loading.

* Enzyme Mixture—0.13 ul 20 mg/ml BSA (in 50% glycerol; BoehringerMannheim), 8 U AMV reverse transcriptase (Seikagaku), 0.2 U E. coliRNase H (Pharmacia), 40 U T7 RNA Polymerase (Pharmacia) and 3 UThermoSequenase (Amersham Life Sciences, Cleveland, Ohio).

When ready for loading and observing detectable reaction products, thereaction products are mixed with an equal volume of STOP/Loading buffer(Formamide, colored dye) and mixed well. 1.5 ul of the resulting mix isloaded per lane of a MicroCel™ gel electrophoresis cassette loaded in aMicroGene Blaster™ DNA sequencer. The sample is electrophoresed anddetected by the automated laser detection system. Results are stored ina computer and analyzed by GeneObjects™ Software (Visible Genetics Inc.,Toronto, Canada).

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO: 1 <211> LENGTH: 29<212> TYPE: DNA <213> ORGANISM: Human immunodeficiency virus type  #1<220> FEATURE: <223> OTHER INFORMATION: sequencing primer for gag # gene<400> SEQUENCE: 1 agtgggggga catcaagcag ccatgcaaa         #                   #            29 <210> SEQ ID NO: 2 <211> LENGTH: 54<212> TYPE: DNA <213> ORGANISM: Human immunodeficiency virus type  #1<220> FEATURE: <223> OTHER INFORMATION: sequencing primer for gag# gene with RNA       polymerase promoter <400> SEQUENCE: 2aattctaata cgactcacta tagggtgcta tgtcacttcc ccttggttct ct#ca           54

What is claimed is:
 1. A method of determining the sequence ofnucleotides in a target ribonucleic acid under isothermal conditionscomprising the steps of: (a) combining in a single reaction vessel anRNA sample containing a target ribonucleic acid, first and secondpolynucleotide primers, wherein the first primer specifically hybridizeswith a target sequence near the 3′ end of the target ribonucleic acid,and the second primer specifically hybridizes to the 3′ end of anantisense copy of the target ribonucleic acid, and wherein at least thefirst or second primer has a detectable label, and wherein at least oneof the first or second primer has an RNA polymerase transcriptioninitiation signal at its 5′ end which signal does not specificallyhybridize to the target ribonucleic acid, ribonucleotide triphosphatesfor RNA synthesis, deoxyribonucleotide triphosphates for DNA synthesis,at least one type of chain terminating nucleotide triphosphate, andenzymes with the activity of reverse transcriptase, RNase H, RNAPolymerase and a low discrimination DNA Polymerase having reduceddiscrimination between dNTP and ddNTP substrates; (b) incubating thecombined reactants under isothermal conditions for a length of timesufficient to generate chain-termninated reaction products, and (c)detecting chain termninated reaction products after electrophoreticseparation, said chain terminated reaction products reflecting thesequence of nucleotides in the target ribonucleic acid.
 2. The method ofclaim 1, wherein the low discrimination DNA polymerase incorporatesdideoxynucleotides into an extending nucleic acid polymer at a ratewhich is no less than about 0.4 times the rate of incorporation ofdeoxynucleocides.
 3. The method according to claim 1, wherein at leastone enzyme having both reverse transctiptase and RNase H activity isincluded in the reaction mixture.
 4. The method according to claim 3,wherein the enzyme having both reverse transcriptase and RNase Hactivity is included in the reaction mixture is selected from amongavian myeloblastosis virus (AMV) reverse transcriptase and Moloneymurine leukemia virus (MMLV) reverse transcriptase.
 5. The methodaccording to claim 1, wherein the deoxynucleotide triphosphates and thechain terminating nucleotide triphosphate are present in a mole ratio offrom 1:300 to 5000:1.
 6. A kit for isothermal sequencing of a target RNAmolecule comprising, in packaged combination, (a) a first polynucleotideprimer of 5-50 nt length which is capable of specific hybridization witha target sequence near the 3′ end of the target RNA molecule (b) asecond polynucleotide primer of 5-50 nt length which is capable ofspecific hybridization with an anti-sense copy of the target RNAmolecule; and (c) enzymes sufficient to provide reverse transcriptaseactivity, RNAse H activity, RNA Polymerase activity and lowdiscrimination DNA polymerase activity having reduced discriminationbetween dNTP and ddNTP substrates, wherein at least one of the first andsecond primers is labeled with a detectable label and wherein at leastone of the first and second primers includes an RNA polymeraseinitiation signal at the 5′-end thereof, which signal does notspecifically hybridize with the target RNA molecule.
 7. The kitaccording to claim 6, wherein the low discrimination DNA polymeraseincorporates dideoxynucleotides into an extending nucleic acid polymerat a rate which is no less than about 0.4 times the rate ofincorporation of deoxynucleotides.
 8. The kit according to claim 6,wherein at least one enzyme having both reverse transcriptase and RNaseH activity is included in the kit.
 9. The kit according to claim 6,wherein at least one of the first or second primers is labeled with afluorescent label.
 10. The method of claim 2, wherein at least oneenzyme having both reverse transcriptase and Rnase H activity isincluded in the reaction mixture.
 11. The method of claim 2, wherein thedeoxynucleotide triphosphates and the chain terminating nucleotidetriphosphate are present in a mole ratio of from 1:300 to 5000:1. 12.The method of claim 3, wherein the deoxynucleotide triphosphates and thechain terminating nucleotide triphosphate are present in a mole ratio offrom 1:300 to 5000:1.
 13. The method of claim 4, wherein thedeoxynucleotide triphosphates and the chain terminating nucleotidetriphosphate are present in a mole ratio of from 1:300 to 5000:1. 14.The kit of claim 7, wherein at least one enzyme having both reversetranscriptase and RNase H activity is included in the kit.
 15. The kitof claim 7, wherein at least one of the first or second primers islabeled with a fluorescent label.
 16. The kit of claim 8, wherein atleast one of the first or second primers is labeled with a fluorescentlabel.